5-Hydroxytryptamine(2A) (serotonin(2A), 5-HT(2A)) receptors are important for many physiologic processes including platelet aggregation, smooth muscle contraction, and the modulation of mood and perception. A large number of pharmaceutical agents mediate their actions, at least in part, by modulating the number and/or activity of 5-HT(2A) receptors. Drugs with action at 5-HT(2A) receptors are used in the treatment of many disorders, including schizophrenia, depression, and anxiety disorders. This review summarizes over two decades of research on the regulation of 5-HT(2A) receptors and provides a comprehensive review of numerous in vivo studies describing the paradoxical phenomenon of 5-HT(2A) receptor down-regulation by chronic treatment with antidepressants and antipsychotics. In addition, studies reporting antagonist-induced internalization of 5-HT(2A) receptors and other G protein-coupled receptors will be highlighted as a possible mechanism to explain this paradoxical down-regulation. Finally, a review of the cellular and molecular mechanisms that may be responsible for agonist-mediated desensitization and internalization of 5-HT(2A) receptors will be presented.

Understanding the precise structure and function of the intracellular domains of G protein-coupled receptors is essential for understanding how receptors are regulated, and how they transduce their signals from the extracellular milieu to intracellular sites. To understand better the structure and function of the intracellular domain of the 5-hydroxytryptamine2A (5-HT2A) receptor, a model G(alpha)q-coupled receptor, we overexpressed and purified to homogeneity the entire third intracellular loop (i3) of the 5-HT2A receptor, a region previously implicated in G-protein coupling. Circular dichroism spectroscopy of the purified i3 protein was consistent with alpha-helical and beta-loop, -turn, and -sheet structure. Using random peptide phage libraries, we identified several arrestin-like sequences as i3-interacting peptides. We subsequently found that all three known arrestins (beta-arrestin, arrestin-3, and visual arrestin) bound specifically to fusion proteins encoding the i3 loop of the 5-HT(2A) receptor. Competition binding studies with synthetic and recombinant peptides showed that the middle portion of the i3 loop, and not the extreme N and C termini, was likely to be involved in i3-arrestin interactions. Dual-label immunofluorescence confocal microscopic studies of rat cortex indicated that many cortical pyramidal neurons coexpressed arrestins (beta-arrestin or arrestin-3) and 5-HT2A receptors, particularly in intracellular vesicles. Our results demonstrate (a) that the i3 loop of the 5-HT2A receptor represents a structurally ordered domain composed of alpha-helical and beta-loop, -turn, and -sheet regions, (b) that this loop interacts with arrestins in vitro, and is hence active, and (c) that arrestins are colocalized with 5-HT2A receptors in vivo.

5-HT(2A) serotonin receptors represent the principal molecular targets for LSD-like hallucinogens and atypical antipsychotic drugs. It has been proposed that a dysregulation of 5-HT(2A) receptor-mediated signaling may contribute to the pathogenesis of schizophrenia and related diseases. A major mechanism for the attenuation of GPCR signaling following agonist activation typically involves the phosphorylation of serine and/or threonine residues by various kinases. Ser/Thr phosphorylation leads to the binding of accessory proteins and the uncoupling of the G proteins, thereby preventing further signaling. The molecular mechanisms by which 5-HT(2A) receptors are desensitized are unknown, and to date, no residues essential for agonist-mediated desensitization have been identified. Thus, we mutated, individually or in groups, all of the 37 serines and threonines in the cytoplasmic domains of the 5-HT(2A) receptor and assessed the effects of these mutations on agonist-mediated desensitization. We discovered that mutation of two residues, S421 in the C-terminal tail and S188 in the second intracellular loop, to alanine resulted in a significant block of agonist-induced desensitization. Intriguingly, a single-nucleotide polymorphism, of unreported frequency, at the S421 locus has been reported (S421F); the S421F mutation, like the S421A mutation, significantly attenuated agonist-mediated desensitization. Taken together, these findings indicate that the process of agonist-mediated desensitization of 5-HT(2A) receptors requires the presence of two nonconserved serine residues located in distinct intracellular loops.

The serotonin (5-hydroxytryptamine) 2A receptor (5-HT2A) is an important G protein-coupled receptor (GPCR) that mediates the effects of hallucinogens and is the target of a number of commonly prescribed medications including atypical antipsychotics, antidepressants, and anxiolytics. The 5-HT2A receptor possesses a canonical Type I PDZ-binding domain (X-Ser/Thr-X-Phi) at the carboxyl terminus and has been predicted, but never demonstrated, to interact with PDZ domain-containing proteins. We discovered that PSD-95, a prototypic PDZ domain-containing protein, directly associates with the 5-HT2A receptor and regulates 5-HT2A receptor-mediated signaling and trafficking in HEK-293 cells. Co-immunoprecipitation studies revealed that the native 5-HT2A receptor, but not a mutant lacking the PDZ-binding domain, interacted directly with PSD-95. The association with PSD-95 enhanced 5-HT2A receptor-mediated signal transduction, a novel action of PSD-95 on GPCRs. The augmentation of 5-HT2A receptor signaling by PSD-95 was not accompanied by alteration in the kinetics of 5-HT2A receptor desensitization but was associated with the inhibition of agonist-induced 5-HT2A receptor internalization. Additional studies demonstrated that 5-HT2A receptor and PSD-95 were co-localized in clusters on the cell surface of HEK-293 cells. Taken together, the present work elucidates novel roles for PSD-95 in regulating the functional activity and intracellular trafficking of 5-HT2A receptors and possibly other GPCRs.

Although a plurality of drugs target G-protein-coupled receptors (GPCRs), most have emerged from classical medicinal chemistry and pharmacology programs and resemble one another structurally and functionally. Though effective, these drugs are often promiscuous. With the realization that GPCRs signal via multiple pathways, and with the emergence of crystal structures for this family of proteins, there is an opportunity to target GPCRs with new chemotypes and confer new signaling modalities. We consider structure-based and physical screening methods that have led to the discovery of new reagents, focusing particularly on the former. We illustrate their use against previously untargeted or orphan GPCRs, against allosteric sites, and against classical orthosteric sites that selectively activate one downstream pathway over others. The ligands that emerge are often chemically novel, which can lead to new biological effects.

5-HT(2A) serotonin receptors are unusual among G-protein coupled receptors in that they can be internalized and desensitized, in some cell types, in an arrestin-independent manner. The molecular basis of the arrestin-insensitivity of 5-HT(2A) receptors is unknown but is probably caused, in part, by the apparent lack of agonist-induced 5-HT(2A) receptor phosphorylation. Because the arrestin-insensitivity of 5-HT(2A) receptors is cell-type selective, we used a "constitutively active" arrestin mutant that can interact with agonist-activated but nonphosphorylated receptors. We show here that this "constitutively active" arrestin mutant (Arr2-R169E) can force 5-HT(2A) receptors to be regulated by arrestins. Cotransfection of 5-HT(2A) receptors with Arr2-R169E induced agonist-independent 5-HT(2A) receptor internalization, and a constitutive translocation of the Arr2-R169E mutant to the plasma membrane, whereas wild-type Arrestin-2 had no effect. Additionally, Arr2-R169E, unlike wild-type arrestin-2, induced a significant decrease in efficacy of agonist-induced phosphoinositide hydrolysis with an unexpected increase in agonist potency. Radioligand binding assays demonstrated that the fraction of receptors in the high-affinity agonist binding-state increased with expression of Arr2-R169E, indicating that Arr2-R169E stabilizes the agonist-high affinity state of the 5-HT(2A) receptor (R*). Intriguingly, the agonist-independent interaction of Arr2-R169E with 5-HT(2A) receptors was inhibited by inverse agonist treatment and is thus probably caused by the high level of 5-HT(2A) receptor constitutive activity. This is the first demonstration that a constitutively active arrestin mutant can both induce agonist-independent internalization and stabilize the agonist-high affinity state of an arrestin-insensitive G protein coupled receptor.

Psychedelics are serotonin 2A receptor agonists that can lead to profound changes in perception, cognition and mood. In this review, we focus on the basic neurobiology underlying the action of psychedelic drugs. We first discuss chemistry, highlighting the diversity of psychoactive molecules and the principles that govern their potency and pharmacokinetics. We describe the roles of serotonin receptors and their downstream molecular signaling pathways, emphasizing key elements for drug discovery. We consider the impact of psychedelics on neuronal spiking dynamics in several cortical and subcortical regions, along with transcriptional changes and sustained effects on structural plasticity. Finally, we summarize neuroimaging results that pinpoint effects on association cortices and thalamocortical functional connectivity, which inform current theories of psychedelic action. By synthesizing knowledge across the chemical, molecular, neuronal, and network levels, we hope to provide an integrative perspective on the neural mechanisms responsible for the acute and enduring effects of psychedelics on behavior.

5-Hydroxytryptamine 2A (5-HT2A) receptors, a major site of action of clozapine and other atypical antipsychotic medications, are, paradoxically, internalized in vitro and in vivo by antagonists and agonists. The mechanisms responsible for this paradoxical regulation of 5-HT2A receptors are unknown. In this study, the arrestin and dynamin dependences of agonist- and antagonist-mediated internalization were investigated in live cells using green fluorescent protein (GFP)-tagged 5-HT2A receptors (SR2-GFP). Preliminary experiments indicated that GFP tagging of 5-HT2A receptors had no effect on either the binding affinities of several ligands or agonist efficacy. Likewise, both the native receptor and SR2-GFP were internalized via endosomes in vitro. Experiments with a dynamin dominant-negative mutant (dynamin K44A) demonstrated that both agonist- and antagonist-induced internalization were dynamin-dependent. By contrast, both the agonist- and antagonist-induced internalization of SR2-GFP were insensitive to three different arrestin (Arr) dominant-negative mutants (Arr-2 V53D, Arr-2-(319-418), and Arr-3-(284-409)). Interestingly, 5-HT2A receptor activation by agonists, but not antagonists, induced greater Arr-3 than Arr-2 translocation to the plasma membrane. Importantly, the agonist-induced internalization of 5-HT2A receptors was accompanied by differential sorting of Arr-2, Arr-3, and 5-HT2A receptors into distinct plasma membrane and intracellular compartments. The agonist-induced redistribution of Arr-2 and Arr-3 into intracellular vesicles and plasma membrane compartments distinct from those involved in 5-HT2A receptor internalization implies novel roles for Arr-2 and Arr-3 independent of 5-HT2A receptor internalization and desensitization.

Dopamine is a neurotransmitter that has been implicated in processes as diverse as reward, addiction, control of coordinated movement, metabolism and hormonal secretion. Correspondingly, dysregulation of the dopaminergic system has been implicated in diseases such as schizophrenia, Parkinson's disease, depression, attention deficit hyperactivity disorder, and nausea and vomiting. The actions of dopamine are mediated by a family of five G-protein-coupled receptors. The D2 dopamine receptor (DRD2) is the primary target for both typical and atypical antipsychotic drugs, and for drugs used to treat Parkinson's disease. Unfortunately, many drugs that target DRD2 cause serious and potentially life-threatening side effects due to promiscuous activities against related receptors. Accordingly, a molecular understanding of the structure and function of DRD2 could provide a template for the design of safer and more effective medications. Here we report the crystal structure of DRD2 in complex with the widely prescribed atypical antipsychotic drug risperidone. The DRD2-risperidone structure reveals an unexpected mode of antipsychotic drug binding to dopamine receptors, and highlights structural determinants that are essential for the actions of risperidone and related drugs at DRD2.